The existence of an adaptive immune system in the lamprey, a jawless basal vertebrate, was posited 40 years ago based on histological analysis and classical immune challenge experiments. Many comparative immunologists believed that lampreys must possess immunoglobulins because mammals employ them in their immune response. However, evidence for immunoglobulins was evasive. Finally, in 2004, Max Cooper and colleagues discovered that the lamprey lack immunoglobulins but rather employ an altogether different receptor species as an antibody. This lamprey antibody, known as the variable lymphocyte receptor (VLR) uses leucine rich repeat motifs to make its receptor face. Analogous to the immunoglobulin system, though probably not because of common ancestry, the VLRs also utilize a genetic recombination mechanism to generate vast somatic antibody diversity. This finding was unexpected, given the ancient natural history of the lamprey and its accepted use by comparative biologists as a primitive vertebrate outgroup. This finding has led to intense interest in the immunological aspects of the VLR system. Despite its basal position, the lamprey has proven to be a valuable repository of new information as much for its differences as for its similarities. To that extent, we have recently discovered that: (1) VLR plays a key role in early embryonic development in addition to its role as an antibody receptor; (2) VLR is involved in providing the cellular microenvironment for building the immune system that subsequently yields the cells that express these antibodies; and (3) programmed genome rearrangements restructure the VLR locus during embryogenesis. The Specific Aims of our proposal are focused around these three observations and take advantage of the complementary expertise of the two PIs, the tractability of the lamprey for experimental work, and the large infrastructure that has been created at Caltech for its functional study. We propose an integrated set of experiments that further define the developmental role of VLRs and elucidate the early VLR repertoire and logic by which the embryonic genomic rearrangements occur at the VLR locus. Our overarching goal in this line of investigation is to understand the biological strategies that allow the amalgamation of complex basic processes that, in this case, involve both immune protection and development. The three independent Aims are inextricably linked by aspects of the VLR system and comprise a mixture of necessary fundamental, as well as more venturesome, experiments. PUBLIC HEALTH RELEVANCE: The emergence of adaptive immunity is considered a major innovation in the origin of the vertebrates, and efforts to understand its genetic underpinnings and molecular mechanisms have been at the forefront of biomedical research and vaccinology for the last few decades. The variable lymphocyte receptor (VLR) is a novel modular receptor system that has recently been discovered in a lineage representing an early offshoot in vertebrate evolution. Its role in adaptive immunity has been shown to be analogous to that of immunoglobulins, and recent literature has referred to VLRs as antibodies despite that fact that they are structurally very different than immunoglobulins. We now have strong evidence that the VLRs are additionally involved in building the early embryo, a completely different role than its antibody function and one that may seem paradoxical for several reasons, particularly with regard to self/non-self recognition and the functional biological limits inherent in a highly diversified receptor system. At a fundamental level, it is of interest to know how such a modular receptor system could have emerged, how it functions, and how it can be molded for deployment in vastly different biological contexts. The VLRs are also of biotechnological [and possibly clinical] relevance since the molecules can be manipulated to encompass many of the same features as conventional monoclonal antibodies.